Spray dry method for encapsulation of biological moieties and chemicals in polymers cross-linked by multivalent ions for controlled release applications

ABSTRACT

Microencapsulation of bioactive and chemical cargo in a stable, cross-linked polymer matrix is presented that results in small particle sizes and is easily scaled-up for industrial applications. A formulation of a salt of an acid soluble multivalent ion, an acid neutralized with a volatile base and one or more monomers that cross-link in the presence of multivalent ions is atomized into droplets. Cross-linking is achieved upon atomization where the volatile base is vaporized resulting in a reduction of the pH of the formulation and the temporal release of multivalent ions from the salt that cross-link the monomers forming a capsule. The incorporation of additional polymers or hydrophobic compounds in the formulation allows control of hydration properties of the particles to control the release of the encapsulated compounds. The operational parameters can also be controlled to affect capsule properties such as particle-size and particle-size distribution.

This application is a 35 U.S.C. §111(a) continuation of PCTinternational application number PCT/US2012/071447 filed on Dec. 21,2012, incorporated herein by reference in its entirety, which claimspriority to, and the benefit of, U.S. provisional patent applicationSer. No. 61/579,893 filed on Dec. 23, 2011, incorporated herein byreference in its entirety. Priority is claimed to each of the foregoingapplications.

The above-referenced PCT international application was published as PCTInternational Publication No. WO 2013/096883 on Jun. 27, 2013, and isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

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INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

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BACKGROUND OF THE INVENTION

1. Field of Invention

This invention pertains generally to the production and use ofmicrocapsules and more particularly to a method for producing smallcross-linked microcapsules in a single step by spray drying, whereinpolymer gelation occurs during spray drying upon volatilization of abase and rapid release of otherwise unavailable multivalent ions as thepH is reduced. A range of small to large microcapsules can be produced.

2. Background

Encapsulation of bioactive moieties is a common practice in the food,biotechnology and pharmaceutical industries to increase the stabilityand shelf life of the encapsulated compound and to control its delivery.In general, the encapsulation matrix confers a protective layer againstadverse environmental conditions and regulates the release of theencapsulated compound in the target application.

Polymers are typically used as the encapsulating medium which allowscross-linking between the molecules to improve overall stability of theencapsulated product. One example is the use of a charged polymer as theencapsulation matrix such that multiple polymers are cross-linked viaelectrostatic interactions with multivalent ions. This form ofion-mediated cross-linking occurs spontaneously upon contact betweenpolymer and ions, and rapidly converts a low-viscosity solution to agelled mass.

Among encapsulation materials, alginates are preferred because of beingnon-toxic, biocompatible and relatively inexpensive. Alginic acids(alginates) are negatively charged polysaccharides readily cross-linkedby divalent calcium ions and ubiquitously utilized in biotechnology andfood applications. Chemically, alginates are linear copolymers of [1→4]linked β-D-mannuronic acid (M) and α-L-guluronic acid (G), arranged asblocks of either type or as a random distribution of each type. They aregenerally obtained from marine brown algae and have varied chemicalstructure and composition depending on the source and harvesting season.An important property of alginates is that they can selectively bindmultivalent cations (e.g. Ca²⁺, Ba²⁺, Zn²⁺, and Al³⁺) in a gentle andalmost temperature independent manner. This gentle solution to geltransition in the presence of selected cations makes alginates an idealimmobilization matrix.

One conventional encapsulation method of forming cross-linked alginatebeads involves dissolving or dispersing the bioactive compound, cells orchemical in an alginate solution and promoting cross-linking bydispersing it into a solution containing the cross-linking agent, knownas the diffusion setting or external gelation method. However, directmixing of alginate and multivalent cations rarely produces homogeneousgels due to the very rapid binding kinetics of such ions. The result isa gel or beads with the highest cross-linked alginate concentrations atthe outer surface with a decreasing gradient of cross-linking towardsthe center of the gel. A different approach known as internal gelationmixes alginates with a cross-linking agent (generally Ca²⁺) in acomplexed or unavailable form and the cation becomes available as the pHchanges. This method is generally accompanied by emulsion and vigorouslystirring, or by introducing the cross-linking agent using a crystal gun.In any case, both encapsulation methods are costly, not easily scaled-upand generally limit the particle size to ≧300 μm. Overall, currentmethods for producing stable alginate gels that involve droppingalginate suspensions into divalent cation solutions are difficult toscale-up and produce undesirably large alginate beads.

In contrast, spray drying is a relatively inexpensive and easilyscaled-up technique that is reproducible and one of the most commonlyused encapsulation methods in industrial settings. The traditional sprayencapsulation process involves dissolving or dispersing the active agentin a sodium alginate solution, forcing the solution through an orificeto form a droplet which is then cross-linked by contact with a calciumchloride solution. Effective spray-drying relies on pumping alow-viscosity solution through an atomizer which has historicallyprecluded ion-mediated cross-linking.

State of art methods for encapsulating biological molecules, cells andchemicals in cross-linked alginates include variations on methods toextrude droplets of alginate/target specie solution into a calciumsolution and are limited in the size of the produced particles such thatonly large (millimeter range) diameters can be achieved. The formationof small (micron-scale), stable particles by spray drying has not beenpractical due to rapid gelation of alginate upon contact with divalentcations. This process has been limited to producing particles largerthan 500 μm.

Microcapsules can contain many different types of materials and can beused for both therapeutic and non-therapeutic applications. Intherapeutic applications, the size of the microcapsules can be animportant factor in the delivery of the capsules across cell membranesas well as the response made by the cell to the microcapsules. It hasbeen shown that smaller microcapsules approximately 300 μm or less tendto avoid a significant cellular inflammatory or immune response and canefficiently cross membranes compared with larger microcapsules. Thereare many other uses for microcapsules that are smaller than the 500 μmlimits of traditional spray drying methods in a wide variety ofapplications.

Accordingly, there is a need for methods for efficiently producing smallmicrocapsules with reproducible characteristics that is inexpensive andcan be scaled up for industrial applications. The present inventionsatisfies these needs as well as others and is generally an improvementover the art.

SUMMARY OF THE INVENTION

The present invention generally provides methods for the production anduse of microcapsules prepared with a single polymerization step viaspray drying of a formulation of a cargo for encapsulation, at least oneacid, at least one volatile base, a salt of an acid soluble multivalention and at least one type of monomer/polymer. In the preferred method,cross-linking of the polymer is achieved by internal gelation that takesplace during spray drying thereby enclosing the cargo in a microcapsule.Ion mediated cross-linking of the polymer molecules is initiallyprevented by pH control with the volatile base. The timing of thecross-linking is also controlled by the timing of the volatilization ofthe base, which lowers the pH and releases the ions to spontaneouslyform cross-links between the polymer molecules.

The methods are particularly useful for spray-drying applications wherepremature cross-linking of the polymers prevents effective atomizationof the product. In the spray-drying application, the pH of a formulationthat includes polymer molecules, an acid and a salt of a divalent ion iscontrolled with a volatile base such that the divalent ions are madeavailable only post-atomization and upon vaporization of the base.Additionally, the release behavior (rates and extent) of theencapsulated moieties in aqueous suspensions can be further controlledby the incorporation of (a) hydrophobic compound(s) in the spray-dryingformulation to control the particle hydration properties.

The methods are illustrated with the spray-drying of an aqueousformulation that contains sodium alginate, a calcium salt that is onlysoluble at reduced pH and an organic acid that has been neutralized to apH just above the pKa with a volatile base. Under these conditions, thecalcium salt is insoluble and calcium ions are not available forcross-linking. The solution in this fluid state is pumped through thenozzle of the spray dryer, where it is effectively atomized. Uponatomization, the volatile base is vaporized, which reduces the pH(hydrogen ions are released into solution) and in turn releases calciumions from the calcium salt that are now available to cross-link thealginate. The incorporation of an additional hydrophobic polymer to theformulation allows for the control of hydration properties of theparticles to control the release of the encapsulated compounds.

This same process can be used for encapsulation using soy or wheyproteins, polygalacturonates (pectins) and other polymers that willspontaneously cross-link in the presence of multivalent cations. Theformulation can also be a mixture of matrix polymer types includingmixtures of polymers and proteins and copolymers. Formulations withmixed polymer types can improve protection of the biological compoundthat is being encapsulated.

The terms “monomer” and “polymer” are used interchangeably herein in thegeneral sense to refer to the molecular entity or unit that has at leastone polymerizable moiety that cross-links in the presence of amultivalent ion and the terms are not intended to be limiting. Thepolymerized unit may be a singular monomer or may be oligomeric. Anymolecule that polymerizes with multivalent ions is a candidatemonomer/polymer.

Preferred volatile bases include ammonium hydroxide and volatile aminecompounds such as methylamine. While these volatile bases are preferred,it will be understood that many different types of volatile bases couldbe selected.

The modulation of the pH of the formulation is by the acid orcombination of acids that are selected. The preferred acid is an organicacid. An anti-oxidative acid can also be used instead of, or as asupplement to, the organic acid in the formulation to increaseprotection for oxygen-sensitive biocompounds in one embodiment.

The preferred salts of multivalent ions that initiate polymerization aredivalent ions such as Ca²⁺, Ba²⁺, and Zn²⁺. Dicalcium phosphate isparticularly preferred. See Table 1.

The formulation can also include other functional compounds such ashydrophobic compounds that can influence release rates of the cargo fromthe microcapsule. The formulation can also include surface-activecompounds to influence particle size and size distributions.

Although the methods are generally directed at employing conventionalspray-drying to encapsulate active agents in matrix particles, otherencapsulating systems can also be used to produce core-shellmicrocapsules. For example, various bi-fluid annular nozzles can beused. Likewise, other coating processes such as spinning disc systemscan be used.

The present methods have been shown to produce stable cross-linkedalginate particles in a single step by spray drying. This approachyields small (median particle sizes in the range of 15-120 μm),insoluble particles whose production could be easily scaled-up forindustrial applications. The operational parameters can also becontrolled to affect product properties such as particle-size andparticle-size distribution.

Furthermore, the activity of the encapsulated bioactives was notcompromised by the methods. Cellulolytic enzymes (cellulases andxylanases) were encapsulated and tested on their different substrates toverify retention of activity. There was no difference in enzyme activitybetween the encapsulated enzymes and equivalent enzyme loadings that didnot undergo spray drying. This demonstrated that enzymes encapsulated bythis spray-drying method can retain full activity. The controlledrelease of encapsulated ingredients was also demonstrated.

According to one aspect of the invention, a one-step process is providedthat includes almost simultaneous particle formation, particlecross-linking and particle drying.

Another aspect of the invention is to provide a method for a)controlling the timing of calcium-mediated cross-linking of polymerssuch as alginates and soy or whey proteins to enable spray-dryencapsulation of biological moieties, b) controlling the release of theencapsulated ingredients by controlling cross-linking and hydrationproperties of the encapsulation, and c) increasing yields ofcross-linked alginate (or soy or whey protein) particles and controllingparticle size and narrowing size distribution.

Yet another aspect of the invention is to provide a microencapsulationmethod that can be adapted for use with many different types ofbiocompatible polymers such as alginate, soy protein, whey protein,chitosan, and pectins.

A further aspect of the invention is to provide a method that can bemodulated to control the microcapsule structural characteristics andproduce capsules of consistent sizes that is easy to use and inexpensiveto produce.

Another aspect of the invention is to provide a method that can be usedin multiple industries employing spray-drying to encapsulate activeagents such as encapsulating biological molecules, cells, probiotics,nutraceuticals and other organic or inorganic chemicals.

Further aspects of the invention will be brought out in the followingportions of the specification, wherein the detailed description is forthe purpose of fully disclosing preferred embodiments of the inventionwithout placing limitations thereon.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood by reference to thefollowing drawings which are for illustrative purposes only:

FIG. 1 is a flow diagram of a method for producing microcapsules using asingle spray-drying step according to one embodiment of the invention.

FIG. 2 is a graph plotting shear stress versus shear rate measurementsof the supernatant of spray-dried particle suspensions stirredvigorously in water overnight.

FIG. 3 is a graph plotting viscosity in the supernatant of spray-driedalginate suspensions over time.

FIG. 4 is a graph plotting size distribution of spray-dried particles asmeasured by Mie Scattering in oil.

FIG. 5 is a graph showing enzyme (protein) concentration in thesupernatant of suspensions of encapsulated enzyme particles.

FIG. 6 is a graph of filter paper activity (FPA) of enzymes from liquidand spray-dried sources.

FIG. 7 is a graph showing xylanase activity in liquid and spray driedenzymes.

FIG. 8 is a graph plotting BSA protein release from cross-linkedalginate encapsulation in aqueous suspensions.

DETAILED DESCRIPTION OF THE INVENTION

Referring more specifically to the drawings, for illustrative purposesseveral embodiments of the materials and methods for producing a rangeof small microcapsules containing selected cargo in a one step spraydrying method of the present invention are depicted generally in FIG. 1through FIG. 8. It will be appreciated that the methods may vary as tothe specific steps and sequence and the microcapsule architecture mayvary as to composition and structural details, without departing fromthe basic concepts as disclosed herein. The method steps are merelyexemplary of the order that these steps may occur. The steps may occurin any order that is desired, such that it still performs the goals ofthe claimed invention.

Methods for microencapsulation of cargo in a stable, cross-linkedpolymer matrix are provided utilizing spray-drying polymerization in asingle polymerization step. The methods consistently produce smallcapsule sizes and the characteristics of the capsule can be controlled.Cargo, that is selected by the user and contained in the core or matrixof the microcapsule, may be exposed to the exterior in some embodimentsbecause the shell is permeable to allow the controlled release of theencapsulated cargo. The permeability of capsule also allows theinteraction of the encapsulated cargo with the surrounding environment.

An alginate encapsulation of a protein cargo is used to illustrate themethod. In this method, an aqueous formulation that contains sodiumalginate, a calcium salt that is only soluble at reduced pH and anorganic acid that has been neutralized to a pH just above the pKa with avolatile base. Calcium ions needed for cross-linking become availableduring spray drying by volatilization of the volatile base and theconsequent drop in the pH of the spraying solution permittingcross-linking of the alginate polymer.

By way of example, and not of limitation, FIG. 1 illustratesschematically a method 10 for producing microcapsules from a formulationthat is aerosolized into droplets through the use of an annular nozzle,spinning disc technology or some other fine droplet forming device suchas spray-drying.

At block 12 a cargo is selected for encapsulation within the sizecontrolled microcapsules. A wide variety of cargo can be selected andencapsulated for different uses. For example, the cargo can be organicor inorganic molecule compounds such as proteins, nucleotides, drugs,medicinal compositions, anti-bacterial agents or probiotics for animalor human treatments. The cargo can also be individual cells, such asstem cells, for implantation. The cargo can also be selected fornon-therapeutic uses such as aromatic compounds, oils, catalysts,initiators and other industrially relevant compounds.

A monomer, polymer or other unit that can be cross-linked in thepresence of multivalent ions is selected at block 14. The selection ofthe cargo and the ultimate use of the microcapsules will influence theselection of the polymer or mixture of polymers/monomers that are usedat block 14. For example, if the ultimate use of the microcapsules isintended to be as an implant in the human body, the selection of thepolymer/monomer at block 14 would be one that is biocompatible as wellas one that does not trigger a significant immune or inflammationresponse. If the ultimate use of the microcapsules is intended as partof a tablet to be ingested, then a polymer can be selected at block 14that will produce a microcapsule that is resistant to degradation in lowpH environments and changing environments.

Cross-linked alginates have been shown to remain mostly intact ingastric environments (i.e. in the stomach) while dissolving in theintestines. Thus compounds that are encapsulated by cross-linkedalginates remain protected in the acid environment of the stomach andonly released once in the intestines. This is advantageous because 1)absorption (of nutrients etc) largely occurs in the large intestines,and 2) this can protect compounds that are otherwise acid labile.

Monomers that are selected at block 14 need to cross-link in thepresence of multivalent ions but not necessarily at low pH conditions.In the embodiment of the invention shown in FIG. 1, the lower pHconditions makes the multivalent ions available for cross-linking thatare unavailable at a higher pH. The invention is not necessarily limitedto low pH conditions, only the availability of multivalent ions.

Suitable polymers can include organic polymers and proteins such asalginate, chitosan, collagen, latex, polygalacturonates (pectins), soyand whey proteins. The capsule polymer matrix can also be formed from amixture of such polymers (e.g. alginates and proteins). The polymersselected at block 14 can also be a combination of polymers andco-polymers such as acrylic latex. Formulations with mixed polymer typescan improve protection of the encapsulated biological compound.

Once the polymer is selected at block 14, at least one acid is selectedat block 16, at least one volatile base is selected at block 18, and thesalt of a multivalent ion is selected at block 20. The salt of themultivalent ion that is selected at block 20 is preferably only solubleat a reduced pH and the selected organic acid or acids will beneutralized to a pH just above the pKa with the selected volatile base.Additionally, Table 1 and Table 2 provide partial lists of calcium saltsand acids, respectively, which can be selected at blocks 16 and 20.Although calcium salts are illustrated, it will be understood that othersalts of multivalent ions can also be selected.

Of the multivalent ions that are capable of cross-linking monomers,divalent ions and trivalent ions are particularly preferred. Any salt ofa divalent or trivalent ion that is soluble only under acidic conditionscan be selected at block 20 and used. For example, salts of barium(Ba²⁺), beryllium (Be²⁺), calcium (Ca²⁺), chromium (Cr²⁺), cobalt(Co²⁺), copper (Cu²⁺), iron (Fe²⁺), lead (Pb²⁺), magnesium (Mg²⁺),mercury (Hg²⁺), strontium (Sr²⁺), tin (Sn²⁺), and zinc (Zn²⁺) can beused. However, dicalcium phosphate, calcium carbonate, calcium oxalateare particularly preferred.

The acid that is selected at block 16 is preferably matched with thevolatile base selected at block 18 so that cross-linking will occur withthe monomers with the volatilization of the base. In one embodiment, ananti-oxidative acid is used instead of or in combination with theorganic acid in the formulation to increase protection foroxygen-sensitive biocompounds. The capsule in this setting has thepotential for exhibiting the anti-oxidative properties of theformulation.

Suitable acids that are selected at block 16 include carboxylic acidssuch as succinic acid and adipic acid, and phenolic acids such as,ascorbic acid, gallic acid and caffeic acid. The acid in the formulationis preferably an acid with a pK in the 4 to 5.5 range.

The volatile base that is selected at block 18 includes ammoniahydroxide, and other volatile amines such as hydrazine, methylamine,trimethylamine, ethylamine, diethylamine, triethylamine, isobutylamine,N,N-diisopropylethylamine, morpholine, piperazine, and ethylenediamine.

At block 22, the selected acids, bases, salts and cargo are mixedtogether to produce a formulation to be atomized and spray dried. Thequantities of each component of the composition are determined by the pHof the resulting formulation and can be optimized. The formulation musthave a pH that maintains the selected multivalent salt as an insolublesalt until liberation by the volatilization of the base. In one otherembodiment, the components of the final formulation are divided andmixed at the nozzle head at the time of atomization at block 24.

The formulation is preferably atomized in a spray dryer or spinning discdryer or other device that will produce droplets of a desired diameterat block 24. The operational parameters of the apparatus can becontrolled to affect capsule properties such as particle-size andparticle-size distribution. The spray dryer preferably produces capsulesin the range of between approximately 15 μm and approximately 400 μm indiameter and capsule diameters ranging from approximately 15 μm toapproximately 120 μm are particularly preferred.

The droplets that are formed in the spray drying apparatus are heated tofurther volatilize the volatile base in the formulation at block 26 toinitiate the polymerization of the polymers and the formation of thecapsules. The volatilization of the base changes the pH of theformulation allowing the salt to disassociate so that multivalent ionsare available for cross-linking of the monomers.

In another embodiment, an annular nozzle is used at block 24 that has across-section consisting of two concentric circles where the corecontaining the biocompound or chemical cargo flows in the inner circlewhile the shell formulation flows from the outer ring. The resultingdroplet from the nozzle will have the biological compound or chemicalcargo in the core of the droplet, while the encapsulating polymer fullysurrounds the core, forming a shell. The droplets are preferably formedin the drying chamber of the apparatus where the volatile base is drivenoff at block 26, releasing the divalent cations and cross-linking thenegatively charged polymers of the shell.

For high melting solid particles, in another embodiment, theencapsulation process takes place utilizing spinning disc technology.The solid particles of the cargo are first dispersed in the alginatesolution containing all the other required ingredients and then droppedonto the spinning disc where the alginate solution coats particlesundergoing centrifugal motion. The coating is then solidified bydropping the coated particles through a heated fluidized bed where theammonia and water are flashed off.

One particularly preferred illustration of the method is with aformulation composed of alginates, adipic acid (pKa=4.43 and 5.41 at 25°C.) or succinic acid (pKa=4.16 and 5.61 at 25° C.) and a calcium salt oflow solubility in water, where the pH is controlled by the addition ofammonium hydroxide. Alginates are mixed polysaccharides of β1→4mannuronic acid and β1→4 guluronic acids. In a solution with a pH >4,the polysaccharide backbone is negatively charged, with pKa's of 4 and3.2 for the mannuronic acid and guluronic acid residues, respectively.The ammonium hydroxide base is titrated to adjust the pH of the solutionto above the second pKa of the acid, thus minimizing the hydrogen ionconcentration in the solution and maintaining the calcium as aninsoluble salt (i.e. not available for cross-linking). The solution inthis fluid state is preferably pumped through the nozzle of the spraydryer, where it is effectively atomized. Upon atomization, the volatileammonia is vaporized which reduces the pH (hydrogen ions are releasedinto solution) and in turn releases Ca²⁺ ions that electrostaticallycross-link the negatively charged backbones of the alginates.

For example, in one embodiment an alginic acid (alginate) solution (4%)can be used that contains alginic acid, unavailable calcium ions in theform of dicalcium phosphate, and citrate, a chelating agent to complexlow concentrations of calcium ions. Biomolecules or other molecules tobe encapsulated are added to the alginic acid solution.

An adipic acid or succinic acid solution (4%) is neutralized to a pHthat is adjusted to above its second pKa (5.4 or 5.6, respectively)using ammonium hydroxide. The alginic acid solution and the neutralizedacid solution are mixed to a ratio of 1:1. No cross-linking will occurwith the mixing. The mixed solution is then spray dried. During spraydrying, the ammonia is volatilized which lowers the pH of the solutionand releases the calcium ions and cross-links the alginates duringatomization. The biomolecules (or other added molecules) are entrappedwithin the alginate matrix.

Optionally, hydrophobic compounds such as latex or waxes can be added tothe formulation before spray drying. In an alternative embodiment,release characteristics of the encapsulated moieties in aqueoussuspensions can be controlled by the incorporation of hydrophobiccompounds in the spray drying formulation. The presence of hydrophobiccompounds in the formulation producing the spray dried product modulatesthe hydration properties of the capsules, thus controlling the rate ofwater intrusion into the capsules and the diffusion of encapsulates outof the particles. Examples of optional hydrophobic compounds in theformulation include (but are not limited to) polymer latexes and waxemulsions.

Finally, the microcapsules are collected at block 28 and may be subjectto further processing such as further curing or may receive additionalcoatings depending on the ultimate use of the microcapsules. Themicrocapsules may also may be separated according to size.

The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed as in any sense limiting the scope ofthe present invention as defined in the claims appended hereto.

Example 1

In order to demonstrate the functionality of the methods, alginates wereused as the matrix for encapsulating a mixture of plant cell walldegrading enzymes. Spray-dried sample and controls that are described inthis example are provided in Table 3.

The microcapsule samples set forth in Table 3 were prepared from thefollowing formulations: Control A consisted of 50 mL of a 2% solution ofManugel® L98 (FMC) in purified water and Control B consisted of 50 mL ofa 1:1 mixture of 4% Manugel® L98 in water and a 4% adipic acid solutionwith pH taken to 5.5 by the addition of a volume of 29% ammoniumhydroxide solution. The sample identified as Example 1 in Table 3 wasmade from 50 mL of a same mixture as Control B with the addition of 48mg of an enzyme mixture consisting of Celluclast®, Novo 188® and NS50030(Novozymes) in a 2:1:1 ratio. Control C consisted of 50 mL of a 1:1mixture of 4% Manugel® L98 in water with a 4% adipic acid solution. Allsolutions were mixed before atomization. Spray drier Model B-290 (BUCHI)was used in the experiments. All atomizations were performed at maximumair flow, 10% pump intensity, 78% aspirator intensity and 150° C. inlettemperature. In all cases, all the volume was pumped into the nozzle andthe recovered spray-dried product was weighed to estimate mass recovery.

Effective cross-linking of the alginates during spray drying wasevidenced by (1) minimal dissolution of the cross-linked alginateparticles in water and (2) the larger average sizes of the cross-linkedalginate particles than the noncross-linked alginate particles. Theextent of the dissolution of each of the alginates was assessed bymeasuring the viscosities of the supernatants of aqueous suspensions ofthe spray-dried particles.

The resistance of cross-linked alginates to dissolution was verified bymeasuring the viscosities of the supernatant in aqueous suspensions ofthe spray-dried particles. The non-cross-linked (Control A) and thecross-linked particles (Control B, Control C and Example 1) (50 mg) wereadded to 5 mL of water and stirred vigorously overnight. The alginatesuspensions were centrifuged for 4 minutes at 3452×g and thesupernatants were collected. The viscosity of the supernatants wasmeasured in a Brookfield DV-II+Pro cone and plate viscometer (BrookfieldEngineering). Alginate suspensions stirred for one-hour and 4-days gavesimilar results. The shear stress versus shear rate measurements of thesupernatant of spray-dried suspensions are shown in FIG. 2 and in Table4.

The viscosity in the supernatant of spray-dried alginate suspensionsover time is shown in FIG. 3. The same alginate-based mass ofnon-cross-linked (NCA) and cross-linked (CA1) alginate particles weresuspended in water (2.5% w/v) and the viscosity of the supernatant wasrecorded over time. Viscosities were obtained from the slopes of shearstress vs. shear rate curves.

The stability of the cross-linking of the alginates in the spray-driedparticles was evaluated by measuring the viscosities of the supernatantof suspended particles over the course of 45 hours. FIG. 3 shows thatthe non cross-linked alginate particles (NCA) dissolved rapidly inwater, resulting in a highly viscous solution (8 mPa·s) within 2 hours.In contrast, the cross-linked alginate particles (CA1) did not dissolveand their supernatant viscosity remained close to unity throughout the45-hour incubation in the aqueous suspension (FIG. 3). Highly stablealginate particles are advantageous in several biomedical applicationssuch as cell immobilization (i.e. stem cells or probiotics) where cellentrapment is crucial for increasing their survival, facilitating theirdelivery and conferring protection from immune responses.

In other samples, the spray-dried particles from a formulationcontaining alginates but no calcium (NCA) and a formulation not designedto avail calcium during atomization (NCM) gave supernatant viscositiesof 9.3 mPa·s and 3.8 mPa·s, respectively. These viscosities,significantly higher than that of water (0.9 mPa·s), indicated that thespray-dried particles readily dissolved in water. Conversely, theviscosities of the supernatants of spray-dried particles that wereformulated to promote alginate cross-linking during spray drying wereonly slightly higher than that of water (1.2-1.3 mPa·s), indicatingminimal dissolution of these particles in water.

Increasing the alginate: Ca2⁺ mass ratio in the formulation resulted inhigher supernatant viscosity (3.3 mPa·s) suggesting insufficientcross-linking in that sample. The amount of cross-linking agent, besidesother factors such as molecular weight of the alginates, the G/M ratioand the block structure of the alginate source, will affect the extentof cross-linking. In another sample, no effort was made to preventgelling prior to spraying, which resulted in a significantly decreasedyield of 20% (mass recovery) as compared to up to 67% from all otherformulations due to difficulties in pumping and atomization.

Overall, the data given in Table 4, FIG. 2 and FIG. 3 demonstrate thatsignificant dissolution of the alginates occurred in the suspension ofthe non-cross-linked alginate particles (Control A) as evidenced by asignificant increase in the viscosity of the supernatant. In contrast,the supernatant of the suspensions containing cross-linked particles(Control B, Control C and Example 1) maintained low viscosities, thusindicating limited dissolution of the matrix.

Example 2

Particle size distributions of the spray-dried particles were analyzedby Mie scattering in the Mastersizer 2000 Particle Size Analyzer(Malvern). The capsule particles were dispersed in ATLOX 4912 (apolymeric surfactant) in corn oil. Capsule sizing was conducted in anorganic phase to minimize the effects of particle swelling that wouldoccur in an aqueous phase, thus more accurately representing the actualsize of the spray-dried particles. The size distribution data fornon-cross-linked alginates and cross-linked alginates (with and withoutencapsulated enzymes) are shown in FIG. 4.

It is interesting to note in FIG. 4 and Table 5, that the medianparticle sizes are generally larger (by approximately 5 to 10 times) forthe cross-linked particles than for the non-cross linked particles. Thisobservation was consistent regardless of the timing of the cross-linking(before or after atomization). The median particle size forenzyme-encapsulated particles was 23 μm. Cross-linked particles appearto be more polydisperse, with a bimodal particle size profile.

A shift towards larger particle sizes was observed in all of the testedcross-linked alginate samples as compared to the non-cross-linkedsamples in addition to those illustrated in FIG. 4. The median size ofnon-cross-linked alginate particles was approximately 5 μm with mostcapsule particles within 2.3 μm and 10.4 μm diameter sizes. In contrast,tested cross-linked alginate particles were more polydisperse, withmedian diameters ranging from 15 to 120 μm depending on the sampleformulation.

This variation in particle size is likely the result of differences insolute concentrations, solution viscosity, rates of volatilization ofammonia and rates of cross-linking during spraying that impact dropletformation and drying kinetics. Solutes alter surface tension and vaporpressure of a solution to impact droplet formation in the spray and thusparticle size in the dried product, with smaller particles obtained atlower solute concentrations. Solute concentrations will also impact thesolution viscosity, thus affecting the size of the droplets formedduring atomization. A surfactant in the solution would lower surfacetension and improve drying kinetics to control for small particle sizes.Solute concentrations in the cross-linked alginate samples were higherthan in the non cross-linked samples due to the addition of the organicacid and ammonium hydroxide.

Example 3

To evaluate enzyme release from the encapsulation matrix over time,microcapsules were produced with a cargo of a cellulase-xylanase mixturein an alginate capsule. Sodium alginate samples were prepared bycompletely dissolving sodium alginate (4% or 2% w/v) in an aqueoussolution containing citric acid (0.06%) and dicalcium phosphate (0.2%)and (in some cases) latex at various concentrations, and then mixing 1:1by volume with succinic acid (4% w/v, pH 5.6 adjusted using ammoniumhydroxide).

The enzyme cargo was a mixture of Celluclast®, Novo 188® and NS 50030(Novozymes NS) that was mixed in a 2:1:1.8 ratio by volume. One volumeof the enzyme mixture was diluted by mixing with three volumes of sodiumacetate 5 mM (pH 5) containing 0.02% sodium azide as preservative andconcentrated in an stirred cell (Amicon) with a 10 kDa MWCO membrane(Millipore).

Enzyme diffusion tests were performed where encapsulated enzymes weresuspended under continuous agitation in an aqueous buffer (5 mM sodiumacetate, pH 5) and sampled over time and the results shown in FIG. 5.The concentration of protein in the final enzyme mixture was measured bythe Bradford assay (Biorad), where increasing protein concentration inthe supernatant indicated an increased diffusion of enzyme into the bulksolution from the encapsulated particles. The data in FIG. 5demonstrates that mass transfer is rapid and the bulk of the diffusionoccurs within the first 5-hours. The diffusion of enzymes out of theencapsulated particles, therefore, is not limited.

The activity of the encapsulated enzymes was also evaluated. Theactivity of the enzyme mixture in the liquid and spray-dried forms wasthen compared and no loss in cellulase and xylanase activity wasobserved as shown in FIG. 6 and FIG. 7.

Cellulase activity was measured on Whatman No. 1 filter paper byincubating the original enzyme mixture and the supernatant of asuspension of spray-dried particles for 1 hour at 50° C. in 5 mM sodiumacetate buffer.

Xylanase activity was similarly measured using birchwood xylan assubstrate instead of filter paper and incubating for 15 minutes.Enzymatic reactions were carried out using freshly spray-dried particlesas well as particles that had been stored for up to 1 month, to verifythere was no loss of activity upon storage.

Activities of the spray-dry encapsulated enzymes were tested againstactivities of the free enzymes in solution on the basis of equivalentprotein loadings. Reactions were carried out in 5 mM sodium acetate pH 5with 0.02% sodium azide in 400 mL total volume at 50° C. Incubationtimes were 60 minutes and 15 minutes for filter paper activity (FPA) andxylanase activity, respectively. Cellulase and xylanase activities wereestimated by measuring the amount of reducing sugars produced in eachreaction. Released sugars were quantified by the dinitrosalicylic (DNS)assay using glucose and xylose standard curves, respectively. Controlreactions using spray-dried powder (with no encapsulated enzyme) plusequivalent concentrations of free enzymes in solution were conducted toverify that other factors in the supernatant of spray-dried particles(i.e. dissolved solids or free alginates) were not interfering with thereaction. It was observed that all the enzyme mixtures tested containedthe same ratios of Celluclast, Novozyme188 and the NS50030 on a volumebasis.

Example 4

To further demonstrate the encapsulation methods, alginates were used asa matrix for encapsulating bovine serum albumin (BSA) with the inclusionof latex molecules in the spray-drying formulation to retard the releaseof encapsulated BSA in aqueous suspensions. Spray-dried samplesdescribed in this example are given in Table 6. Samples were prepared asfollows: CA1±BSA consisted of a 1:1 mixture of 2% solution of sodiumalginate (Sigma-Aldrich cat. no A2158) sodium citrate (0.06%) anddicalcium phosphate (0.2%) in water and a 4% succinic acid solution withpH taken to 5.6 by addition of a 29% ammonium hydroxide solution.CA1L0.05±BSA consists of a 1:1 mixture of 1% solution of sodiumalginate, citric acid (0.06%) and dicalcium phosphate (0.2%) and latex(0.05%) in water and a 4% succinic acid solution with pH taken to 5.6 byaddition of a 29% ammonium hydroxide solution. The same formulation isprepared for CA1L0.25±BSA and CA10.5±BSA with the exception of 0.25% and0.5% latex loading, respectively, instead of 0.05% latex loading. Forall samples, the ‘±BSA’ indicates that samples with and without BSA wereprepared, where 0.15% (w/v) of BSA was added to the +BSA formulationshortly before spray drying. All solutions were mixed beforeatomization. Spray drier Model B-290 (BUCHI) was used in theexperiments. All atomizations were performed at maximum air flow, 20%pump intensity, 100% aspirator intensity and 150° C. inlet temperature.The latex used was chosen for its glass transition temperature of 75°C., which is lower than the outlet temperatures of approximately 80° C.In all cases, all the volume was pumped through the nozzle.

During spray-drying, volatilization of the ammonia from the atomizeddroplet reduces the solution pH to approximately 4.2 to 4.4 (the firstpKa of the organic acid present in the formulation). In this pH range,BSA has a net-positive charge (pI=4.7), thus facilitating strongattractive electrostatic interactions with the negatively chargedcarboxyl groups of the alginates.

The cargo was a standard BSA solution (0.15% w/v in H₂O; ThermoScientific) that was added to the spray-drying solution right beforespray drying. Moisture content of the spray-dried samples was measuredin a Mettler Toledo HR83 halogen moisture analyzer, followingmanufacturer guidelines and using three replicates per sample. Thespray-dried samples were stored in a desiccator with anhydrous calciumsulphate (Hammond Drierite Company, Xenia, Ohio).

Time-course protein diffusion from cross-linked alginate particles intothe liquid phase was obtained by taking 250 mg of the spray-dried powderand adding 5 mL 0.02% NaN₃ (aqueous solution) and mixing thoroughly.Samples were centrifuged (1 min at 562×g) at different time points and100 mL aliquots of the supernatant were collected. Protein in thesupernatant was quantified using the Pierce BCA protein assay using BSAstandards.

Spray-dried samples without protein but otherwise with identicalformulations were used as a control. The extent of protein diffusion outof the spray-dried particles was determined as the total mass of theprotein measured in the supernatant as a percent of the total proteinadded. No interference of non-cross-linked alginates and/or latex in thesupernatant with the protein quantitation assay was noted, verified bymeasuring a known amount of BSA added to control samples.

Example 5

The release of encapsulated BSA in aqueous suspensions was evaluated todemonstrate the permeability of the microcapsules. BSA release fromcross-linked alginate particles into the liquid phase was obtained bymixing 250 mg of the spray-dried powder and 5 mL 0.02% NaN₃ (aqueoussolution). Samples were centrifuged (1 min at 562×g) at several timepoints and 100 μL aliquots of the supernatant were collected. Protein inthe supernatant was quantified using the Pierce BCA protein assay thatis based on the 2,2′-bicinchoninate method using BSA standards.Spray-dried samples without protein but with otherwise identicalformulations were used as a control.

In this illustration, varying amounts of hydrophobic styrene acryliclatex was added to the formulations to affect protein release from thespray-dried particles. The latex that was used was chosen based on itsglass transition temperature (Tg) of 75° C., slightly below the outlettemperature during spray drying (80° C.) to promote softening duringspraying and fusing during drying in the particles.

The extent of protein diffusion out of the spray-dried particles wasdetermined as the total mass of the protein measured in the supernatantas a percent of the total protein added. No interference of noncross-linked alginates and/or latex in the supernatant with the proteinquantitation assay was observed, verified by measuring a known amount ofBSA added to control samples. Results shown in FIG. 8 demonstrate thatincreasing levels of latex in the spray-dry formulation decreased BSArelease rates and the extent of release (with the 70 hours that weretested).

Release characteristics of the encapsulated BSA were examined bymeasuring the rate of protein diffusion out of the particles intoaqueous media. Only up to approximately 65% of the added protein wasmeasured in the liquid phase for any of the samples tested as seen inFIG. 8. Furthermore, incubation for an extended 3-day period with 1%(v/v) Triton X-100 and brief sonication did not increase the amount ofprotein released from the particles. In addition to measuring totalnitrogen in the spray dried samples, several different commerciallyavailable protein assays (Bradford Protein Assay (Bio-Rad), BCA ProteinAssay (Pierce) and Quant-iT Protein Assay (Invitrogen)), were used toattempt to measure the amount of protein encapsulated in the spray-driedparticles.

A relatively fast initial release rate of the encapsulated BSA wasobserved as seen in FIG. 8 that may be due to large pores in thecross-linked alginate beads or to BSA being at the surface of theparticles. BSA is a surface-active protein that will preferentiallypartition to the air/water interface during droplet formation, and ithas been observed that BSA adsorption at the air/water interface is arapid process. Addition of low-molecular weight surfactants that wouldout-compete larger molecules such as proteins for the air/waterinterface would prevent the rapid release of the encapsulated compound.

There were notable differences in release rates between the differentformulations in the first 6 hours, where higher latex contents appear tocorrelate with lower rates and extents of release as seen in FIG. 8. Thelowest amount of latex added (0.05 g latex/g alginates, CA1L0.05) hadminimal impact on BSA release. Increasing latex content in theformulation trended with decreasing total recovery of BSA in thesupernatant. A linear fit of the BSA recovered in solution with respectto latex content yields an R² value of 0.94 (not shown). The hydrophobiclatex incorporated into the encapsulation matrix may inhibit waterdiffusion into the particles, thereby limiting the release of BSA. Theaddition of water insoluble substances to the alginate matrix was shownto modify the surface porosity and improve entrapment of theencapsulated moieties.

Example 6

Manipulation of the capsule size and capsule permeability were alsodemonstrated. It was observed that the addition of BSA to theformulations impacted the size and shape of the spray-dried particles.BSA-containing capsules were smaller and had a narrower particle sizedistribution compared to those capsules without BSA but with anotherwise identical formulation. Additionally, the BSA-loaded particleshad more regular and homogeneous shapes. BSA is a globular,surface-active protein that likely decreased surface tension in thespray, thus resulting in smaller droplets with faster drying kinetics.Furthermore, BSA-containing formulations resulted in increased yields ona mass basis with less sample lost in the cyclone, possibly due to theimproved drying. In contrast, the formulation containing thecellulase/xylanase mixture yielded larger, more polydisperse particles.

In addition, components other than the enzymes present in thepreparation (such as sorbitol) may result in an increase in theviscosity and a decrease in the vapor pressure of the spraying solutionresulting in larger droplets, slower drying and lower mass recovery.

Moreover, further optimization of spray-drying conditions could yieldeven smaller particles. For example, increasing air pressure through theatomizer, or the use of alternate nozzle configurations could improvespray atomization and decrease particles size. Alternatively, the feedrate has a direct influence on the outlet temperature.

Normally it is preferred that the feed rate be minimal to allow for gooddrying of the particles with an outlet temperature between approximately72° C. and 80° C. Increasing the feed rate will decrease the outlettemperature and can result in microcapsules that insufficiently driedcompromising recovery and increasing apparent particle size due toaggregation in the collection vessel. The feed rate can be optimized forparticular polymers, acids, bases salts and cargo. Mechanical parametersin the spray dryer can also be adjusted to further control particle sizeand aggregation.

The addition of surface-active compounds to the liquid feed such as BSAresulted in smaller particles with a narrower size distribution, likelydue to improved drying kinetics. Particle size and shape can be furthercontrolled by modifying the liquid feed formulation along withspray-drying parameters. Alginate gelation by single step spray dryingwas stable in aqueous suspensions. This was in contrast tonon-cross-linked particles obtained with the same spray-dryingparameters, which rapidly dissolved in the same aqueous solution.

About 65% of the encapsulated protein was released to the supernatant inaqueous suspensions of spray-dried particles obtained with the differentformulations tested. Further incubation with a non-ionic surfactant andsonication did not increase the amount of protein released from theseparticles. This low protein recovery was possibly due to electrostaticinteractions between the encapsulated protein and the negative charge inthe alginates backbone and/or to the higher extent of cross-linkingobtained by the internal gelation used in the method (in contrast toexternal gelation). Protein loss during spray drying could not bediscarded. Furthermore, the addition of a hydrophobic polymer to thespraying formulations impacted the release rate of protein from thespray-dried particles, with higher latex concentrations resulting in alower extent of protein release. This was possibly due to restrictedwater diffusion into the particles thereby limiting BSA release.

Additionally, a fast initial protein release rate was also observed.This was possibly due to the large pores generally associated tocross-linked alginates and/or to BSA being at the surface of theparticles. BSA is a surface active protein that has been shown topartition to the air/water interface quite rapidly in spray dryingexperiments.

Accordingly, the present invention provides a method formicroencapsulation of cargo compounds in a stable, cross-linked alginatematrix that results in small particle sizes and is easily scaled-up forindustrial applications. The gentle gelation and moderate chemicalenvironment used in the method will be useful for encapsulating avariety of bioactive compounds including cells, biopolymers andchemicals for many commercial applications, including in the food andpharmaceuticals industries. The methods are easily adapted to specificapplications and can produce capsules with customized particle sizes andshapes as well as release kinetics.

From the discussion above it will be appreciated that the invention canbe embodied in various ways, including the following:

1. A method of cross-linking polymer molecules, comprising: (a) mixingmonomer molecules, at least one salt of an acid soluble multivalent ionand an acid neutralized with a volatile base; and (b) volatilizing saidvolatile base, thereby liberating said multivalent ions and initiatingcross-linking of the monomer molecules.

2. The method as recited in embodiment 1, wherein the monomer isselected from the group of monomers consisting of alginates,polygalacturonates, chitosan, collagen, latex, soy proteins and wheyproteins.

3. The method as recited in any of the previous embodiments, wherein themultivalent ion is a divalent cation.

4. The method as recited in any of the previous embodiments, wherein thedivalent ion is selected from the group of ions consisting of barium(Ba²⁺), calcium (Ca²⁺), chromium (Cr²⁺), copper (Cu²⁺), iron (Fe²⁺),magnesium (Mg²⁺) and zinc (Zn²⁺).

5. The method as recited in any of the previous embodiments, wherein theacid is an organic acid selected from the group of acids consisting ofadipic acid, acrylic acid, glutaric acid, succinic acid, ascorbic acid,gallic acid and caffeic acid.

6. The method as recited in any of the previous embodiments, wherein thevolatile base is selected from the group of volatile bases consisting ofammonia, methylamine, trimethylamine, ethylamine, diethylamine andtriethylamine.

7. A method for producing microcapsules, comprising: (a) providing aformulation comprising: (i) monomer molecules; (ii) at least one acidneutralized with a volatile base; and (iii) an insoluble salt of amultivalent ion; (b) atomizing the formulation to form droplets; and (c)volatilizing the volatile base of the droplets, thereby lowering the pHof the formulation and making available said multivalent ion tocross-link the monomer molecules.

8. The method as recited in embodiment 7, further comprising adding acargo to the formulation prior to atomization.

9. The method as recited in any of the previous embodiments, wherein theformulation further comprises a copolymer.

10. The method as recited in any of the previous embodiments, whereinthe formulation further comprises a hydrophobic compound.

11. The method as recited in any of the previous embodiments, whereinthe hydrophobic compound comprises latex.

12. The method as recited in any of the previous embodiments, whereinthe monomer is selected from the group of monomers consisting ofalginates, polygalacturonates, chitosan, collagen, latex, soy proteinsand whey proteins.

13. The method as recited in any of the previous embodiments, whereinthe salt is selected from the group of salts consisting of dicalciumphosphate, calcium carbonate, calcium oxalate, calcium phosphate,calcium meta-silicate and calcium tartrate.

14. The method as recited in any of the previous embodiments, whereinthe acid is an organic acid selected from the group of acids consistingof adipic acid, acrylic acid, glutaric acid, succinic acid, ascorbicacid, gallic acid and caffeic acid.

15. The method as recited in any of the previous embodiments, whereinthe volatile base is a base selected from the group of bases consistingof ammonia, methylamine, trimethylamine, ethylamine, diethylamine, andtriethylamine.

16. A method for producing microcapsules, comprising: (a) providing aformulation comprising: (i) a plurality of at least one type of monomermolecule; (ii) citrate; (ii) at least one acid neutralized with avolatile base; (iii) a salt of an acid soluble multivalent ion; and (iv)a hydrophobic compound; (b) atomizing said formulation to form droplets;and (c) volatilizing the volatile base of the droplets, thereby loweringthe pH of the formulation and making available the multivalent ion tocross-link the monomer molecules; wherein the hydrophobic compoundmodifies hydration properties of the dried particles to retard releaseof encapsulated compounds.

17. The method as recited in any of the previous embodiments, whereinthe hydrophobic compound comprises a compound selected from the group ofcompounds comprising polymer latexes, wax emulsions and surfactants.

18. The method as recited in any of the previous embodiments, whereinthe monomer is selected from the group of monomers consisting ofalginates, polygalacturonates, chitosan, collagen, latex, soy proteinsand whey proteins.

19. The method as recited in any of the previous embodiments, whereinthe salt is selected from the group of salts consisting of dicalciumphosphate, calcium carbonate, calcium oxalate, calcium phosphate,calcium meta-silicate and calcium tartrate.

20. The method as recited in any of the previous embodiments, whereinthe acid is an organic acid selected from the group of acids consistingof adipic acid, acrylic acid, glutaric acid, succinic acid, ascorbicacid, gallic acid and caffeic acid.

21. A method of cross-linking polymer molecules embodiment for use inspray drying applications to encapsulate biomolecules, cells and otherchemical entities, comprising the steps of (a) providing a formulationcomprising: (i) alginate polymer molecules; (ii) citric acid; (iii)adipic acid; (iv) ammonium hydroxide; and (v) dicalcium phosphate; (b)atomizing the formulation in a spray dryer; and (c) volatilizing theammonia, as a result of the atomizing, thereby making calcium ionsavailable for cross-linking the alginate polymer molecules.

22. A method of cross-linking polymer molecules embodiment for use inspray drying applications to encapsulate biomolecules, cells and otherchemical entities, with control over the release rates of theencapsulated compounds, comprising the steps of (a) providing aformulation comprising: (i) alginate polymer molecules; (ii) citrate;(iii) succinic acid; (iv) ammonium hydroxide; (v) dicalcium phosphate;and (vi) hydrophobic compound; (b) atomizing the formulation in a spraydryer; and (c) volatilizing the ammonia, as a result of the atomizing,thereby making calcium ions available for cross-linking the alginatepolymer molecules while modifying hydration properties of the driedparticles to retard release of the encapsulated compounds.

23. A method of cross-linking polymer molecules embodiment for use inspray drying applications to encapsulate biomolecules, cells and otherchemical entities, and control the release rates of encapsulatedcompounds, comprising the steps of (a) providing a formulationcomprising: (i) alginate polymer molecules; (ii) citric acid; (iii)succinic acid; (iv) ammonium hydroxide; (v) dicalcium phosphate; and(vi) a latex polymer; (b) atomizing the formulation in a spray dryer;and (c) volatilizing the ammonia, as a result of the atomizing, therebymaking calcium ions available for cross-linking the alginate polymermolecules while modifying hydration properties of the dried particles toretard release of the encapsulated compounds.

24. A method of cross-linking polymer molecules embodiment for use inspray drying applications to encapsulate biomolecules, cells and otherchemical entities, and control over release rates of encapsulatedcompounds, comprising the steps of (a) providing a formulationcomprising: (i) alginate polymer molecules; (ii) citrate; (iii) ascorbicacid; (iv) ammonium hydroxide; (v) dicalcium phosphate; and (vi) a latexpolymer; (b) atomizing the formulation in a spray dryer; and (c)volatilizing the ammonia, as a result of the atomizing, thereby makingcalcium ions available for cross-linking the alginate polymer moleculeswhile conferring anti-oxidative properties of the dried particles toprotect oxygen-sensitive encapsulated compounds.

Although the description above contains many details, these should notbe construed as limiting the scope of the invention but as merelyproviding illustrations of some of the presently preferred embodimentsof this invention. Therefore, it will be appreciated that the scope ofthe present invention fully encompasses other embodiments which maybecome obvious to those skilled in the art, and that the scope of thepresent invention is accordingly to be limited by nothing other than theappended claims, in which reference to an element in the singular is notintended to mean “one and only one” unless explicitly so stated, butrather “one or more.” All structural, chemical, and functionalequivalents to the elements of the above-described preferred embodimentthat are known to those of ordinary skill in the art are expresslyincorporated herein by reference and are intended to be encompassed bythe present claims. Moreover, it is not necessary for a device or methodto address each and every problem sought to be solved by the presentinvention, for it to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed under the provisions of 35U.S.C. 112 unless the element is expressly recited using the phrase“means for.”

TABLE 1 Examples Of Suitable Calcium Salts Chemical Chemical Solubilityin H₂O Soluble in Name Formula (g/100 g) Acid? Dicalcium CaH(PO₄)•2H₂O0.02 (24.5° C.) yes phosphate Calcium CaCO₃ 0.015 (25° C.) yes carbonateCalcium CaC₂O₄•H₂O 0.0071 (25° C.) yes oxalate Calcium Ca(PO₄)₂0.002-0.003 (cold) yes phosphate Calcium CaSiO₃ 0.0095 (cold) yesmetasilicate Calcium CaC₄H₄O₆•4H₂O 0.0032 (cold) yes tartrate

TABLE 2 Examples Of Acids That Can Be Used In This Invention Acid pK_(a)Adipic 4.43, 5.41 Succinic 4.16, 5.61 Acrylic 4.25 Glutaric 4.34, 5.41Ascorbic 4.10, 11.6 Gallic 4.5, 10  Caffeic 4.62

TABLE 3 Spray-Dried Sample And Controls Described In Example 1 MassSample Additional Recovery Sample ID type Description comments (%)Control A Non-cross- No acid was added to Solution is easily 67 linkedthe solution prior to pumped through alginate spraying, thus the Ca²⁺nozzle. is never made available to cross-link. Control B Alginates ThepH is controlled as Solution is easily 64-67 cross- described. Cross-pumped through linked linking happens after nozzle. Exhaust post-spraying and during vapor is at high pH, spraying drying of theparticles in verifying that the through the the drying chamber. ammoniais nozzle vaporized during the drying process. Example 1 Alginates Sameas above, but Same comments as 60-64 plus with enzymes above. Enzymesenzyme (cellulases and are encapsulated in cross- xylanases) in the thealginate matrix. linked at solution with the the nozzle encapsulationmatrix. Control C Alginates The pH of the sample This sample was 21cross- was not controlled in mixed and sprayed linked the mannerdescribed quickly before before above and the cross-linking spray-availability of Ca²⁺ for completely drying cross-linking was notprevented spraying. controlled or limited. The spraying nozzle wasquickly clogged and yields were low.

TABLE 4 Linear regression R²-values and viscosities from FIG. 2 Linearregression Viscosity Sample ‘R²’ (μ)mPa*s Control A 0.999 5.53 Control B0.992 1.13 Example 1 0.856 1.80 Control C 0.995 1.11 H₂O 0.986 0.75

TABLE 5 Size Distribution Of Spray-Dried Particles Measured By MieScattering In Oil Particle size (μm) Sample d(0.1) d(0.5) d(0.9) ControlA 2.808 7.372 23.695 Control B 8.079 37.092 218.932 Example 1 6.14622.884 76.529 Control C 4.848 19.234 69.314

TABLE 6 Compositions Of Typical Spray-dry Formulations Sample TypeSample ID^(†) Formulation (%, w/v in H₂O) Non cross- NCA Alginate (2)linked alginates Cross-linked CA1 ± BSA Alginate (1) CaHPO₄•2H₂O (0.1)Na-citrate alginates (0.03) succinic acid^(‡) (2) ± BSA (0.15)Cross-linked CA1L0.05 ± Alginate (1) CaHPO₄•2H₂O (0.1) Na-citratealginates and BSA (0.03) latex (0.05) succinic acid^(‡) (2) ± BSA latex(0.15) Cross-linked CA1L0.25 ± Alginate (1) CaHPO4•2H₂O (0.1) Na-citratealginates and BSA (0.03) latex (0.25) succinic acid^(‡) (2) ± BSA latex(0.15) Cross-linked CA1L0.5 ± Alginate (1) CaHPO₄•2H₂O (0.1) Na-citratealginates and BSA (0.03) latex (0.5) succinic acid^(‡) (2) ± BSA (0.15)latex Cross-linked CA2 ± BSA Alginate (2) CaHPO₄•2H₂O (0.1) Na-citratealginates (0.03) succinic acid^(‡) (2) ± BSA (0.15) Non cross- NCMManugel (2) linked Manugel Cross-linked CM ± BSA Manugel (2) adipicacid^(‡) (2) ± BSA (0.15) Manugel Cross-linked CM ± Manugel (2) adipicacid^(‡) (2) ± Manugel cellulase cellulase/xylanase mixture (0.048)^(†)± BSA indicates that samples with and without bovine serum albuminwere prepared. ^(‡)Prepared separately by dissolving in water andadjusting pH > pK_(a) of the organic acid (≈5.6) with ammoniumhydroxide.

We claim:
 1. A method for producing microcapsules, comprising: (a)providing a formulation comprising: (i) monomer molecules; (ii) at leastone acid neutralized with a volatile base; and (iii) an insoluble saltof a multivalent ion; (b) atomizing said formulation to form droplets;and (c) volatilizing said volatile base of said droplets, therebylowering the pH of the formulation, which dissolves the otherwiseinsoluble salt, thereby making available said multivalent ion tocross-link monomer molecules, forming microcapsules.
 2. The method asrecited in claim 1, further comprising adding a cargo to saidformulation prior to atomization.
 3. The method as recited in claim 1,wherein said formulation further comprises a copolymer.
 4. The method asrecited in claim 1, wherein said formulation further comprises ahydrophobic compound.
 5. The method as recited in claim 4, wherein saidhydrophobic compound comprises latex.
 6. The method as recited in claim1, wherein said monomer is selected from the group of monomersconsisting of alginates, polygalacturonates, chitosan, collagen, soyproteins and whey proteins.
 7. The method as recited in claim 1, whereinsaid insoluble salt is selected from the group of salts consisting ofdicalcium phosphate, calcium carbonate, calcium oxalate, calciumphosphate, calcium meta-silicate and calcium tartrate.
 8. The method asrecited in claim 1, wherein said acid is an organic acid selected fromthe group of acids consisting of adipic acid, acrylic acid, glutaricacid, succinic acid, ascorbic acid, gallic acid and caffeic acid.
 9. Themethod as recited in claim 1, wherein said volatile base is a baseselected from the group of bases consisting of ammonia, methylamine,trimethylamine, ethylamine, diethylamine, and triethylamine.
 10. Amethod for producing microcapsules, comprising: (a) providing aformulation comprising: (i) a plurality of at least one type of monomermolecule; (ii) citrate; (iii) at least one acid neutralized with avolatile base; (iv) a salt of an acid soluble multivalent ion; and (v) ahydrophobic compound; (b) atomizing said formulation to form droplets;and (c) volatilizing said volatile base of said droplets, therebylowering the pH of the formulation, which dissolves the otherwiseinsoluble salt, thereby making available said multivalent ion tocross-link monomer molecules, forming microcapsules; (d) wherein thehydrophobic compound modifies hydration properties of the microcapsulesto retard release of an encapsulated cargo.
 11. The method as recited inclaim 10, wherein said hydrophobic compound comprises a compoundselected from the group of compounds comprising polymer latexes, waxemulsions and surfactants.
 12. The method as recited in claim 10,wherein said monomer is selected from the group of monomers consistingof alginates, polygalacturonates, chitosan, collagen, soy proteins andwhey proteins.
 13. The method as recited in claim 10, wherein said saltis selected from the group of salts consisting of dicalcium phosphate,calcium carbonate, calcium oxalate, calcium phosphate, calciummeta-silicate and calcium tartrate.
 14. The method as recited in claim10, wherein said acid is an organic acid selected from the group ofacids consisting of adipic acid, acrylic acid, glutaric acid, succinicacid, ascorbic acid, gallic acid and caffeic acid.